scholarly journals Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

2013 ◽  
Vol 14 (6) ◽  
pp. 11544-11559 ◽  
Author(s):  
Winnie Kam ◽  
Vanessa Lake ◽  
Connie Banos ◽  
Justin Davies ◽  
Richard Banati
Keyword(s):  
Polymerase Chain Reaction ◽  
Gamma Irradiation ◽  
Nuclear Dna ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Copy ◽  
Chain Reaction ◽  
Copy Numbers ◽  
Polymerase Chain ◽  
Gene Copy Numbers

scholarly journals Utility of Real-Time Quantitative Polymerase Chain Reaction in DetectingMycobacterium tuberculosis

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Zhongquan Lv ◽  
Mingxin Zhang ◽  
Hui Zhang ◽  
Xinxin Lu
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Roc Curve ◽  
Quantitative Polymerase Chain Reaction ◽  
Culture Method ◽  
Gene Copy ◽  
Youden Index ◽  
Chain Reaction ◽  
Cutoff Value ◽  
Polymerase Chain

This study aimed to assess the value of real-time quantitative polymerase chain reaction (RT-qPCR) for the detection ofMycobacterium tuberculosis(MTB). Samples from 192 patients with suspected MTB were examined by RT-qPCR and an improved Löwenstein–Jensen (L-J) culture method. To evaluate the diagnostic usefulness of RT-qPCR in detecting MTB, a receiver operating characteristic (ROC) curve for RT-qPCR was generated, and the area under the curve (AUC) as well as a cutoff value was calculated. Using the L-J culture method as the gold standard, accuracy of the RT-qPCR method for detecting MTB was 92.7%, with sensitivity and specificity of 62.5% and 97.02%, respectively. In comparison with the improved L-J culture method, the AUC of RT-qPCR ROC curve was 0.957, which was statistically significant (p<0.001). The Youden Index reached the maximum value (0.88) for gene copy number of 794.5 IU/mL, which was used as the cutoff value. RT-qPCR detection of MTB yielded results consistent with those of the improved L-J culture method, with high accuracy. RT-qPCR may be used as an auxiliary method for etiological diagnosis of tuberculosis.

scholarly journals Brief communication (Original). Rapid diagnosis of trisomy 21 by relative gene copy using real-time quantitative polymerase chain reaction

2014 ◽  
Vol 8 (3) ◽  
pp. 399-403
Author(s):  
Chinnuwat Sanguansermsri ◽  
Pranoot Tanpaiboon ◽  
Pimlak Charoenkwan ◽  
Arunee Phusua
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Trisomy 21 ◽  
Diagnostic Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Chromosome 21 ◽  
Gene Copy ◽  
Chain Reaction ◽  
Karyotypic Analysis ◽  
Polymerase Chain

Abstract Background: Trisomy 21 or Down syndrome (DS) is the most common aneuploidy disorder. Fetal karyotypic analysis remains the criterion standard for prenatal diagnosis of DS, although the method is time consuming and requires skilled personnel. Real-time quantitative polymerase chain reaction (qPCR) can be used to determine a difference in the amount of gene copy by calculation of the difference between the cycle threshold (ΔCT) of a tested gene and a reference gene. Objectives: To develop a rapid qPCR diagnostic method for trisomy 21. Methods: Ten DS patients with the known karyotype of trisomy 21 were enrolled. Their parents were included as controls. D21S11 locus on chromosome 21 and SM locus on chromosome 16 from each subject were amplified by qPCR. The D21S11/SM ΔCT and 2-ΔΔCT values were compared between DS patients and their parents. Results: The D21S11/SM ΔCT values of the DS patients were higher than their respective controls except for one family. The mean 2-ΔΔCT value between patients and mothers was 1.88 ± 0.95 (95% CI 1.20-2.56), and between fathers and mothers as controls was 1.06 ± 0.68 (95% CI 0.58-1.54). Conclusion: The diagnostic method of trisomy 21 by using qPCR is feasible, although false negative results may occur. Using more index genes is recommended to increase the sensitivity and specificity.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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